Melanija Tomić

The P2X7 Receptor Channel Pore Dilates under Physiological Ion Conditions

Activation of the purinergic P2X7 receptor leads to the rapid opening of an integral ion channel that is permeable to small cations. This is followed by a gradual increase in permeability to fluorescent dyes by integrating the actions of the pannexin-1 channel. Here, we show that during the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak. The secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. The biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and the Δ18 mutant with deleted P2X7 receptor–specific 18–amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents. These results indicate that the P2X7 receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore.

Experimental Characterization and Mathematical Modeling of P2X7 Receptor Channel Gating

The P2X7 receptor is a trimeric channel with three binding sites for ATP, but how the occupancy of these sites affects gating is still not understood. Here we show that naive receptors activated and deactivated monophasically at low and biphasically at higher agonist concentrations. Both phases of response were abolished by application of Az10606120, a P2X7R-specific antagonist. The slow secondary growth of current in the biphasic response coincided temporally with pore dilation. Repetitive stimulation with the same agonist concentration caused sensitization of receptors, which manifested as a progressive increase in the current amplitude, accompanied by a slower deactivation rate. Once a steady level of the secondary current was reached, responses at high agonist concentrations were no longer biphasic but monophasic. Sensitization of receptors was independent of Na+ and Ca2+ influx and ∼30 min washout was needed to reestablish the initial gating properties. T15E- and T15K-P2X7 mutants showed increased sensitivity for agonists, responded with monophasic currents at all agonist concentrations, activated immediately with dilated pores, and deactivated slowly. The complex pattern of gating exhibited by wild-type channels can be accounted for by a Markov state model that includes negative cooperativity of agonist binding to unsensitized receptors caused by the occupancy of one or two binding sites, opening of the channel pore to a low conductance state when two sites are bound, and sensitization with pore dilation to a high conductance state when three sites are occupied.

EFFECTS OF INSULIN-LIKE GROWTH FACTORS I AND II AND INSULIN ON THE IMMORTALIZED HYPOTHALAMIC GTI-7 CELL LINE

Insulin and insulin-like growth factor I (IGF-I) participate in energy metabolism, regulate cellular growth and differentiation, and are thought to act locally in a paracrine manner through specific receptors. Systemic levels of these peptides in humans and primates are directly associated with levels of activity of the reproductive axis. To date, it is unclear whether these peptides participate in reproductive function by acting at the level of the GnRH neuron. In this study we examined the effects of IGF-I, IGF-II and insulin on immortalized GnRH-secreting neurons, the GTI-7 cell line. The GTI-7 cells expressed all three members of the insulin receptor family as determined by analysis of 125I-IGF-I, 125I-IGF-II and 125I-insulin binding sites. Insulin receptors bound insulin, IGF-II and IGF-I with a ratio of potency of 1:5:20. IGF-I and IGF-II receptors bound both IGF-I and IGF-II. The ratio of potency of IGF-I/IGF-II was 1:5 for the IGF-I receptor and 100:1 for the IGF-II receptor. The binding characteristics of the growth factors at 22 degrees C suggested the possibility that these cells may secrete IGF binding proteins. To ensure that changes in GnRH levels in the media were due to secretion and not to changes in cell number, the mitogenic effect of these peptides on GTI cells was evaluated. Both insulin and IGF-I were strong mitogens (48-hour incubation), restoring cell number to that of serum-replete cultures at a dose of 0.1 ng/ml. A 100-fold higher dose of IGF-II was required to produce a similar level of mitogenicity, implicating an action through the IGF-I and/or insulin receptor. Due to these mitogenic effects, the effect of insulin, IGF-I and IGF-II on GnRH secretion was studied after short-term exposure. Insulin and IGF-I did not affect GnRH secretion, but IGF-II had a biphasic effect on GnRH release after 2 h of incubation (a maximum stimulatory effect occurred with a 0.1 ng/ml dose). In order to examine the signal transduction mechanism, the role of cytoplasmic calcium mobilization in IGF-II-induced GnRH secretion was examined in single cells using calcium imaging. The effect of IGF-II on GnRH secretion appeared to operate via a calcium-independent mechanism. The studies document an insulin/IGF system in the GTI-7 neuronal cell line and show that insulin and IGFs can exert direct effects on the immortalized GnRH neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

IDENTIFICATION OF AMINO ACID RESIDUES CONTRIBUTING TO DESENSITIZATION OF THE P2X2 RECEPTOR CHANNEL

The P2X2 receptor (P2X2R) is a member of the ATP-gated ion channels that mediate Ca2+ entry in several tissues, including the brain, adrenal medulla, and pituitary. Alternative usage of cryptic splice sites in the primary P2X2R transcript accounts for the existence of several transcript types, one of which (P2X2–2R) encodes a functional channel. P2X2–2R lacks a stretch of cytoplasmic C-terminal amino acids (Val370-Gln438) and exhibits rapid and complete desensitization, whereas P2X2R desensitizes slowly and incompletely. The role of the C terminus in P2X2R desensitization was studied by generating several channel mutants and monitoring intracellular free Ca2+ changes in transfected single GT1–7 neurons. Deletion studies indicated that the Arg371-Ile391 segment of the P2X2R is required for sustained Ca2+ influx. To identify the important residues within this segment, three contiguous amino acids were sequentially changed to alanine. Only two of these replacement mutants, at Arg371-Thr372-Pro373and Lys374-His375-Pro376, had an enhanced rate of desensitization. Single amino acid deletions in the P2X2R C terminus and a series of insertions of wild-type sequences into the corresponding spliced site identified four residues, Pro373-Lys374-His375-Pro376, required for sustained Ca2+ influx through agonist-occupied wild-type channels. Thus, it is likely that the Pro373-Pro376 sequence of P2X2R represents a functional motif that is critical for the development of the slow desensitization profile observed in these channels. Consequently, deletion of this motif by alternative splicing provides an effective mechanism for generating a channel with controlled Ca2+ influx.

Role of aromatic and charged ectodomain residues in the P2X4 receptor functions

The localization of ATP binding site(s) at P2X receptors and the molecular rearrangements associated with opening and closing of channels are still not well understood. At P2X4 receptor, substitution of the K67, F185, K190, F230, R278, D280, R295, and K313 ectodomain residues with alanine generated low or non‐responsive mutants, whereas the F294A mutant was functional. The loss of receptor function was also observed in K67R, R295K, and K313R mutants, but not in F185W, K190R, F230W, R278K, and D280E mutants. To examine whether the loss of function reflects decreased sensitivity of mutants for ATP, we treated cells with ivermectin, an antiparasitic agent that enhances responsiveness of P2X4R. In the presence of ivermectin, all low or non‐responsive mutants responded to ATP in a dose‐dependent manner, with the EC50 values for ATP of about 1, 2, 4, 20, 60, 125, 270, 420, 1000 and 2300 μmol/L at D280A, R278A, F185A, K190A, R295K, K313R, R295A, K313A, K67A and K67R mutants, respectively. These results indicate that lysines 67 and 313 and arginine 295 play a critical role in forming the proper three‐dimensional structure of P2X4R for agonist binding and/or channel gating.

Local Regulation of Gonadotroph Function by Pituitary Gonadotropin-Releasing Hormone

Cultured rat pituitary cells and immortalized pituitary gonadotrophs (αT3–1 cells) express specific messenger RNA transcripts for GnRH and exhibit positive immunostaining for the GnRH peptide. Each cell type released GnRH during both static culture and perifusion, albeit in lesser amounts than cultured hypothalamic cells and GT1–7 neurons. In perifused pituitary cells, exposure to a GnRH agonist stimulated the release of GnRH as well as LH. In contrast, treatment with a GnRH receptor antagonist or with GnRH antiserum decreased basal LH release. In pituitary cell cultures, a small proportion of gonadotrophs exhibited high amplitude and low frequency baseline Ca2+ oscillations in the absence of GnRH stimulation. Such spontaneous oscillations were comparable to those induced by picomolar concentrations of GnRH and could be abolished by treatment with a GnRH antagonist. These in vitro findings indicate that locally produced GnRH causes low level activation of pituitary GnRH receptors, induces spontaneous intr...

Characterization of a plasma membrane calcium oscillator in rat pituitary somatotrophs.

In excitable cells, oscillations in intracellular free calcium concentrations ([Ca2+] i ) can arise from action-potential-driven Ca2+ influx, and such signals can have either a localized or global form, depending on the coupling of voltage-gated Ca2+ influx to intracellular Ca2+ release pathway. Here we show that rat pituitary somatotrophs generate spontaneous [Ca2+] i oscillations, which rise from fluctuations in the influx of external Ca2+ and propagate within the cytoplasm and nucleus. The addition of caffeine and ryanodine, modulators of ryanodine-receptor channels, and the depletion of intracellular Ca2+ stores by thapsigargin and ionomycin did not affect the global nature of spontaneous [Ca2+] i signals. Bay K 8644, an L-type Ca2+ channel agonist, initiated [Ca2+] i signaling in quiescent cells, increased the amplitude of [Ca2+] i spikes in spontaneously active cells, and stimulated growth hormone secretion in perifused pituitary cells. Nifedipine, a blocker of L-type Ca2+channels, decreased the amplitude of spikes and basal growth hormone secretion, whereas Ni2+, a blocker of T-type Ca2+ channels, abolished spontaneous [Ca2+] i oscillations. Spiking was also abolished by the removal of extracellular Na+ and by the addition of 10 mm Ca2+, Mg2+, or Sr2+, the blockers of cyclic nucleotide-gated channels. Reverse transcriptase-polymerase chain reaction and Southern blot analyses indicated the expression of mRNAs for these channels in mixed pituitary cells and purified somatotrophs. Growth hormone-releasing hormone, an agonist that stimulated cAMP and cGMP productions in a dose-dependent manner, initiated spiking in quiescent cells and increased the frequency of spiking in spontaneously active cells. These results indicate that in somatotrophs a cyclic nucleotide-controlled plasma membrane Ca2+oscillator is capable of generating global Ca2+ signals spontaneously and in response to agonist stimulation. The Ca2+-signaling activity of this oscillator is dependent on voltage-gated Ca2+ influx but not on Ca2+ release from intracellular stores.

Expression and Roles of Pannexins in ATP Release in the Pituitary Gland

Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 in AtT-20 pituitary cells enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland.

Characterization of Novel Pannexin 1 Isoforms from Rat Pituitary Cells and their Association with ATP-gated P2X Channels

Our previous studies have showed that Pannexin 1 (Panx1), a member of a recently discovered family of gap junction proteins, is expressed in the pituitary gland. Here we investigated the presence and expression pattern of Panx1 isoforms in pituitary cells, their roles in ATP release, and their association with purinergic P2X receptor subtypes that are native to pituitary cells. In addition to the full-size Panx1, termed Panx1a, pituitary cells also express two novel shorter isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4, respectively. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.

Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1–2 in hypothalamic and pituitary cells

Hypothalamic and pituitary cells express G protein-coupled adenosine and P2Y receptors and cation-conducting P2X receptor-channels, suggesting that extracellular ATP and other nucleotides may function as autocrine and/or paracrine signaling factors in these cells. Consistent with this hypothesis, we show that cultured normal and immortalized pituitary and hypothalamic cells release ATP under resting conditions. RT-PCR analysis also revealed the presence of transcripts for ecto-nucleotidase eNTPDase 1–2 in these cells. These enzymes were functional as documented by degradation of endogenously released and exogenously added ATP. Blocking the activity of eNTPDases by ARL67156 led to an increase in ATP release in perifused pituitary cells and inhibition of degradation of extracellularly added ATP. Furthermore, the addition of apyrase, a soluble ecto-nucleotidase, and the expression of recombinant mouse eNTPDase-2, enhanced degradation of both endogenously released and exogenously added ATP. The released ATP by resting hypothalamic cells was sufficient to activate and desensitize high-affinity recombinant P2X receptors, whereas facilitation of ATP metabolism by the addition of apyrase protected their desensitization. These results indicate that colocalization of ATP release sites and ecto-nucleotidase activity at the plasma membrane of hypothalamic and pituitary cells provides an effective mechanism for the operation of nucleotides as extracellular signaling molecules.